Sangeeta Kulkarni

Mutations in the gyrA and parC genes of quinolone-resistant Neisseria gonorrhoeae isolates in India

A dramatic increase in the number of quinolone-resistant Neisseria gonorrhoeae isolates in India and worldwide has been reported recently. This study was undertaken to identify and characterise mutations in the gyrA and parC genes of N. gonorrhoeae resistant to six different quinolone antibiotics. In total, 64 N. gonorrhoeae clinical isolates were obtained during 2007-2009 from patients attending sexually transmitted diseases clinics (New Delhi, 35; Pune, 16; Mumbai, 6; Hyderabad, 6; and Nagpur, 1). Antimicrobial susceptibility was determined by Etest and mutation patterns in gyrA and parC were determined by sequencing analysis. All strains showed varying resistance to different quinolone analogues, ranging from 17.2% (gatifloxacin) to 98.4% (ofloxacin and norfloxacin). Sequencing of gyrA and parC revealed that 100% of strains showed mutations in gyrA and 46.9% showed mutations both in gyrA and parC. All strains showed single or double mutations at Ser-91→Phe, Ser-91→Thr and Asp-95→Gly/Asn in gyrA and at Glu-91→Gly in parC. Asp-95→Asn mutation was the most prevalent in strains isolated from New Delhi, whilst Asp-95→Gly was prevalent in strains isolated from Pune. Strains were categorised into eight different mutation patterns. Resistant strains with high minimum inhibitory concentrations (≥8 μg/mL) showed mutations both in gyrA and parC. The difference in the proportion of strains showing mutations in gyrA and parC was found to be significant (P<0.001). The mutation Asp-95→Asn was restricted to Pune strains only. These results indicate that mutations in quinolone target enzymes may have resulted in the high-level resistance seen in these isolates.

Antimicrobial activity of plant extracts against sexually transmitted pathogens

Comprehensive management of sexually transmitted infections (STIs) using vaginal or rectal microbicide-based intervention is one of the strategies for prevention of HIV infection. Herbal products have been used for treating STIs traditionally. Herein, we present in vitro activity of 10 plant extracts and their 34 fractions against three sexually transmitted/reproductive tract pathogens – Neisseria gonorrhoeae, Haemophilus ducreyi and Candida albicans. The plant parts were selected; the extracts/fractions were prepared and screened by disc diffusion method. The minimum inhibitory and minimum cidal concentrations were determined. The qualitative phytochemical analysis of selected extracts/fractions showing activity was performed. Of the extracts/fractions tested, three inhibited C. albicans, ten inhibited N. gonorrhoeae and five inhibited H. ducreyi growth. Our study demonstrated that Terminalia paniculata Roth. extracts/fractions inhibited growth of all three organisms. The ethyl acetate fraction of Syzygium cumini Linn. and Bridelia retusa (L.) Spreng. extracts was found to inhibit N. gonorrhoeae at lowest concentrations.

Antimicrobial susceptibility testing, auxotyping, and serotyping of Neisseria gonorrhoeae strains isolated in India.

Sixty-four Neisseria gonorrhoeae strains isolated from patients attending sexually transmitted disease clinics at Pune and Delhi between January 2007 and June 2008, were subjected to antimicrobial susceptibility testing, auxotyping, and serotyping. We observed 6 antibiotic resistance patterns, 6 auxotypes, 3 serogroups, and 17 serovars. The combination of auxotyping and serotyping is a potential useful method for typing N. gonorrhoeae as a result of high discriminatory index, rapidity, ease, and relatively lower cost.

Performance of tests for identification of Neisseria gonorrhoeae

Sir, The identification of Neisseria gonorrhoeae by sensitive, precise, and rapid technique is important for the correct diagnosis and appropriate treatment of patients with gonorrhoea. For decades, the standard method of identification of the pathogen has been acid production from cystine tryptic agar (CTA) medium containing carbohydrates1. For a more rapid identification of this pathogen a variety of immunological and biochemical commercial kits have now become available. An ideal test should have high sensitivity and specificity, should be easier to perform and cheap. An attempt was made in this study to identify such a test by comparing four available tests on 71 N. gonorrhoeae isolates. The three most widely used immunological kits are the MicroTrak N. gonorrhoeae culture confirmation test (an immunofluoresence test; Trinity Biotech PIC, Ireland), the Phadebact Monoclonal GC test (a coagglutination test; MKL Diagnostics AB, Kung Hans Vag, Sollentuna, Sweden) and GonoGen II (a membrane immunoassay; New Horizons Diagnostics, USA), all of which employ monoclonal antibodies (mAbs) developed against specific epitopes on the two types of the major outer membrane protein, protein I A and B (PIA and PIB)2. Interpretation of the MicroTrak test is less subjective but requires a fluorescence light microscope whereas Phadebact monoclonal test is easier to perform and can be used by most small laboratories2,3. GonoGen II is a colorimetric test and does not require heat treatment step of the coagglutination test. This test eliminates the reading of agglutination; instead, the mixture of antigen-antibody complex is passed through a filtration unit. The appearance of a red dot on the filter indicates a positive reaction2. Though the specificity of these kits for detection of N. gonorrhoeae is high, both false-positive (cross-reactions with other Neisseria species such as N. meningitidis, N. lactamica, N. cinerea and Kingella denitrificans) and false-negative results have also been reported4. The biochemical kits widely used are the Neisseria Preformed Enzyme Test (PET), Gonochek II, RapID NH, Neisstrip, API-NH and Rapid carbohydrate utilization test (RCUT)3,4,5. These tests are based on the presence of preformed chromogenic enzyme in the culture, and thus require a heavy inoculum of the organism grown on selective medium to permit rapid speciation of isolates. The enzymes that are detected by these systems include beta-galactosidase, gamma-glutamylaminopeptidase and prolyl-hydroxyprolyl aminopeptidase2. The Neisseria PET and Gonochek II are both single-use tubes containing chromogenic substrates that detect the presence of three preformed enzymes, each of which is indicative of a pathogenic Neisseria species, namely N. lactamica, N. meningitidis and N. gonorrhoeae. In Gonochek II, after incubation a red colour change (hydrolysis of L-proline 4-methoxynapthylamide) is observed2,3. The API-NH and RapID NH kits employ a battery of tests, combining carbohydrate utilization and preformed enzymes. These two tests detect the change in prolyl iminopeptidase (PIP) enzyme activity due to the mutation in the pip gene. Not all isolates of N. gonorrhoeae possess PIP activity6,7. In RCUT, preformed enzyme is measured by adding a suspension of the overnight growth of the suspect organism to a buffered (non-nutrient) solution containing the sugar to be tested and a pH indicator. A yellow colour change indicated a positive reaction and orange red colour as negative reaction8. As taxonomic differences between members of the Neisseria genus remain small, the identification of this pathogen can be problematic. Of the several commercial kits used worldwide for identification of N. gonorrhoeae, only four commercial kits, RCUT, API-NH, Gonochek II and Phadebact GC system were available in India. The purpose of this study was to compare the performance of these four commercial biochemical methods to identify pathogenic N. gonorrhoeae and to assess the cost effectiveness and utility in the clinical laboratory. A total of 71 Neisseria isolates were included in the study carried out at the National AIDS Research Institute (NARI), Pune, India, during January 2007 to June 2009. Of these, 64 isolates of N. gonorrhoeae were obtained from patients attending STD clinics of NARI, Pune (n=16), Safdarjang hospital, New Delhi (n=35) and 13 collected under STI-Operations Research project conducted at NARI (Mumbai-6, Hyderabad-6 and Nagpur-1). Patients attending STD clinics with complaints of urethral/cervical discharge were included in the study. Seven non gonococcal Neisseria strains, N. sicca (n=2), N. cinerea (n=2) and N. lactamica (n=3) recovered from clinical specimens from patients attending NARI clinics were also included. All the strains were tested by API-NH (Bio Merieux SA, France), Gonochek II (E-Y Laboratories, CA, USA), Phadebact monoclonal GC OMNI test (Boule Diagnostics AB, Sweden) according to manufacturers’ instructions, and by Rcut8. N. gonorrhoeae ATCC 49226 (β-lactamase negative) and WHO strains A, G H, E and O (received under WHO Gonococcal Antimicrobial Surveillance Programme from WHO GASP South East Asia Region Reference Laboratory, New Delhi) (β-lactamase positive) were used as quality control in each test run. The cost per test for each identification method was calculated considering the cost of media, reagents, ancillary supplies and time required to perform identification by each test method. All isolates of N. gonorrhoeae were correctly identified with RCUT system and Phadebact monoclonal GC OMNI system (Table). Gonochek II test correctly identified in 68 (95.3%) of N. gonorrhoeae isolates. The low sensitivity of this test has also been reported earlier. API-NH system correctly identified 51 (79.6%) of N. gonorrhoeae isolates. Thirteen (20.3%) N. gonorrhoeae isolates did not show any reaction for PIP enzyme in the API-NH system, which could be done to the lack of enzyme in these isolates. Specificity of these tests was evaluated based on their ability to give negative results on the seven non gonococcal strains. RCUT system correctly identified N. cinerea, N. sicca strains and one of the three N. lactamica strains as non gonococcal strains. Similar findings were observed by Young and Moyes9. API-NH system correctly identified both N. cinerea strains, one of the two N. sicca strains and one of the three N. lactamica strains as non gonococcal strains. Gonochek II test correctly identified one of the three N. lactamica strains as non gonococcal. Other studies have also reported similar findings in identifying N. gonorrhoeae, N. lactamica and N. sicca strains5,10. The Phadebact monoclonal GC OMNI system correctly identified both N. cinerea strains, two of the three N. lactamica strains and one of the two N. sicca strains. The sensitivity and specificity of Phadebact monoclonal GC OMNI system in our study was similar to that reported earlier5. Thus, RUCT appeared to be the most specific (6/7) followed by Phadebact OMNI system (5/7), and API-NH (4/7). Gonochek II test showed lowest specificity by identifying six of the 7 non gonococcal strains as N. gonorrhoeae. RCUT was found to be the cheapest among all tests (Table). Table Comparison of four test systems for the identification of N. gonorrhoeae isolates The Phadebact monoclonal GC OMNI system was rapid, simple to perform, accurate and provided results in 2-3 min. However, it showed moderate specificity and was more expensive than other methods. The colours generated in the Gonochek II system incubated at 30°C for 30 min were distinct, easy to read, stable and easy to handle in the clinical laboratory. However, it had low sensitivity (95.3%) in identification of N. gonorrhoeae. Additionally, it appeared to have low specificity since six of the seven non gonococcal strains were identified as gonococcal strains. API-NH system showed low sensitivity and specificity in identification of N. gonorrhoeae. This may be due to the lack of enzyme PIP in these isolates which have also been reported in other studies4,5,6,7. We found that the RCUT gave reliable results within 4 h, and identified 100 per cent of N.gonorrhoeae isolates as reported earlier by Yong and Moyes9. Additionally, it appeared to be highly specific since only one of seven non gonococcal strains was wrongly identified as gonococcal strain. Considering the performance, rapidity and cost, RCUT appeared to be the best among the four methods studied for confirmatory identification of N.gonorrhoeae. RCUT can be used routinely as it is cheaper and easily available but the reagents used for RCUT should be evaluated with the known control strain before being used routinely.

Performance of microscopy for detection of microsporidial spores from stool samples of HIV infected individuals with diarrhoea.

Sir, Intestinal microsporidiosis is the most common cause of chronic diarrhoea in treatment-naive HIV infected patients1. Intestinal microsporidiosis due to Encephalitozoon (Septata) intestinalis and Enterocytozoon bieneusi are also most frequently reported among immunocompromised patients such as transplant recipients2–4. Laboratory diagnosis of microsporidiosis was initially based on the detection of microsporidial spores in stool specimens by light microscopy using special stains5. Electron microscopy, wherever possible, was also used for detection of microsporidia6–8. Diagnosis of intestinal microsporidiosis has been markedly improved by the use of Webers chromotrope stain and its modified procedure1,5,9, and fluorescence staining with fluorochromes such as Uvitex 2B or calcoflour white which binds to the chitin in the endospore layer of the microsporidial spore1,8,10,11. Molecular technique such as polymerase chain reaction (PCR) became available later for confirmation of micropsoridia. Additionally, species specific PCR can be used to identify different species of microsporidia12–16. However, due to resource constraints, most laboratories rely on the use of microscopy of stool samples for diagnosis of intestinal microsporidiosis. Limited studies have been carried out in India on detection of microsporidia8,17,18. We compared light microscopy with modified trichome stain, fluorescence microscopy using Uvitex 2B and PCR to detect microsporidia in HIV infected individuals with diarrhoea during January 2009 to May 2010 on consecutive HIV infected patients with diarrhoea admitted at Naidu Municipal Corporation Hospital, Pune. The study protocol was approved by the institutional ethics committee & Naidu Municipal Corporation Hospital. The study included 331 patients [65 (51 males, 14 females)] HIV infected adults >18 yr and 266 HIV (uninfected individuals) with diarrhoea. This study was carried out only on HIV infected individuals with diarrhoea. Study patients were interviewed using the structured questionnaire. Diarrhoea was defined as two or more liquid or three or more soft stools per day All the patients had history of diarrhoea of <14 days and were treatment-naive HIV-infected individuals. Stool samples were collected in wide mouth, leak proof, clean sterile containers and then transported to National AIDS Research Institute (NARI) within 4 h of collection. If there was a delay in the transportation, the samples were preserved at 4o C. The samples were immediately processed after receiving at NARI for microscopy by the conventional method8. Stool samples were stained by modified trichome and Uvitex 2B by the method described earlier4,6,8, and were subjected to light microscopy and fluorescence microscopy, respectively. A portion of the stool was stored at -70°C for further molecular analysis. DNA was extracted from frozen samples using the QIA amplication DNA tissue kit (Qiagen, Inc, Germany). PCR was performed using specific primers as described by Najla et al19. The median duration of diarrhoea was found to be 3 days (IQR 2.75 - 5) and median number of episodes of diarrhoea was noted as 6 in HIV-infected individuals (n=65). The mean age was found to be 35.1 yr ranging from 18-60 yr. Male (n=51) outnumbered the females (n=14). The CD4 counts were available only for 35 patients [CD4 count 200-499 cells/μl (n= 29) and CD4 count ≥500 cells /μl (n=6)] Examination of 65 stool samples from 331 patients by light microscopy with modified trichome stain showed microsporidial spores (oval shaped, measuring 1-2 μm in diameter under oil immersion) in four samples. Fluorescence microscopy using Uvitex 2B also showed microsporidial spores in the same four (6.2%) HIV-infected individuals. PCR detected microsporidial spores in stool samples of five (7.7%) individuals including four found positive by light and fluorescent microscopy (Fig.). Of these five samples positive by PCR, three were identified as Enterocytozoon bieneusi and two were identified as Encephalitozoon intestinalis. A repeat light microscopy and fluorescent microscopy on one sample positive only by PCR did not yield positive results. Thus, the results, though limited by a small number of positives, indicate that trichome and Uvitex 2B stains work well for diagnosis of intestinal microsporidiosis. PCR as expected was more sensitive and yielded one additional positive sample. Fig. (a) Stool smear stained with modified trichome stain showing microsporidial spores (arrow); (b) Stool smear stained by Uvitex 2B & examined with UV light. spores of microsporidia show typical elongated shape (arrow); (c) Agaraose gel electrophoresis ... In conclusion, with well trained laboratory staff both the Uvitex 2B and modified trichome stain can be used for microsporidia detection. PCR can increase sensitivity and species identification. The study was limited by small sample size and small number of positives and hence warrants additional testing on a large number of samples.

Antibiotic susceptibility pattern of Neisseria gonorrhoeae strains isolated from five cities in India during 2013–2016

Purpose. Emergence of multidrug resistance in Neisseria gonorrhoeae, an STI of public health significance is the biggest challenge to gonorrhoea control. Monitoring for antimicrobial resistance is essential for the early detection of emergent drug resistance patterns. Methodology. One hundred and twenty four N. gonorrhoeae strains were isolated between September 2013‐August 2016 [82‐New Delhi, 3‐Pune, 3‐Mumbai, 20‐Secunderabad and 16‐Hyderabad] to determine antimicrobial susceptibility and to compare the CLSI disc diffusion method with Etest for these strains. The results of the two methods were compared by using kappa statistics. Results. Ninety eight percent [CI: 96.2‐100] of isolates were resistant to ciprofloxacin, 52 % [CI: 43.2‐60.8] to penicillin, 56 % [CI: 47.2‐64.7] to tetracycline and 5 % [CI: 1.2‐8.8] to azithromycin. All the strains were susceptible to spectinomycin, ceftriaxone and cefixime except for two strains which showed decreased susceptibility to ceftriaxone and cefixime. Kappa scores for penicillin, azithromycin, ciprofloxacin, ceftriaxone and cefixime showed that the CLSI method had high agreement with Etest while tetracycline had substantial agreement. Conclusion. Our data suggest that the disc diffusion method which is both cost effective and more feasible, can effectively be used routinely for monitoring antibiotic susceptibility in N. gonorrhoeae, in limited resource countries like India. We demonstrate the emergence of decreased susceptibility to ceftriaxone and cefixime and threshold levels of resistance to azithromycin in India. This underscores the importance of maintaining continued surveillance for antibiotic resistance in N. gonorrhoeae and a potential requirement for strategic change in guidelines in the not so distant future.